CN118679387A - Nonspecific reaction inhibitor - Google Patents
Nonspecific reaction inhibitor Download PDFInfo
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- CN118679387A CN118679387A CN202380021890.1A CN202380021890A CN118679387A CN 118679387 A CN118679387 A CN 118679387A CN 202380021890 A CN202380021890 A CN 202380021890A CN 118679387 A CN118679387 A CN 118679387A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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Abstract
本发明提供一种非特异性反应抑制剂，其溶解性高，能够确保充分的添加量，廉价且批次差少。本发明的非特异性反应抑制剂包含肽或核酸分子与高分子化合物的复合物，所述肽或核酸分子与非特异性反应物质特异性结合。
The present invention provides a nonspecific reaction inhibitor, which has high solubility, can ensure sufficient addition amount, is cheap and has little batch difference. The nonspecific reaction inhibitor of the present invention comprises a complex of a peptide or nucleic acid molecule and a polymer compound, wherein the peptide or nucleic acid molecule specifically binds to a nonspecific reaction substance.
Description
Technical Field
The present invention relates to a nonspecific reaction inhibitor for accurately detecting a trace amount of a substance in an immunoassay method and inhibiting a nonspecific reaction that is an obstacle when quantitative determination is performed.
Background
Japanese patent application laid-open No. 11-287801 discloses a method for suppressing a nonspecific reaction by allowing an antibody against a nonspecific reaction substance to coexist in a measurement reagent to reduce the nonspecific reaction by the nonspecific reaction substance in a sample to be examined. However, there are the following problems in this technique: when IgG or IgM is used as an antibody against a nonspecific reaction substance, an immune complex is formed with the nonspecific reaction substance, and an immunoscattering turbidimetric reaction occurs, and thus a measurement by the latex turbidimetry may not show an accurate measurement value. As a countermeasure against this problem, if the amount of IgG and IgM added to the nonspecific reaction substance is reduced, the nonspecific inhibitory effect may not be sufficiently obtained.
In order to eliminate the immune scattering turbidimetric reaction, igG is cleaved into F (ab') 2 to reduce hydrophobicity, so that the IgG can be added to a reagent in a large amount and the immune scattering turbidimetric reaction can be made difficult to occur. However, there is a technical problem in the persistence of the nonspecific inhibitory effect in the reagent, and it is expected that the cause thereof is the decrease in the nonspecific inhibitory effect due to the decomposition of F (ab ') 2 into Fab'. In japanese patent No. 5189098, a method is disclosed in which a high molecular compound is modified on an antibody fragment such as Fab' to thereby increase the size of the molecule, and thus the nonspecific inhibitory effect is enhanced.
Prior art literature
Patent literature
Patent document 1: japanese patent laid-open No. 11-287801
Patent document 2: japanese patent No. 5189098
Disclosure of Invention
First, the technical problem to be solved
However, in recent years, an immunoassay method having higher sensitivity has been demanded, and if the amount of a sample to be used is increased for the purpose of high sensitivity of a reagent, the amount of a nonspecific reaction substance present in a reaction system becomes extremely large, and accordingly, it is necessary to coexist a large amount of a nonspecific reaction inhibitor in the reagent. In addition, when a large amount of a nonspecific substance is present in a sample, it is also necessary to cause a large amount of a nonspecific reaction inhibitor to coexist in a reagent. Under such circumstances, since the technique of japanese patent No. 5189098 uses relatively expensive biological materials such as antibodies and antibody fragments, there are technical problems in terms of cost and batch differences of the materials, and since the nature of the technique is a huge molecular size, the effect of improving the solubility in the reagent is insufficient, and when the sample size is increased for the purpose of high sensitivity, the amount of addition to ensure sufficient non-specific inhibitory effect cannot be satisfied.
In view of the above, an object of the present invention is to provide a nonspecific reaction inhibitor which has high solubility, can ensure a sufficient amount of addition, is inexpensive, and has a small lot size.
(II) technical scheme
In order to achieve the above-described problems, intensive studies have been conducted, and it is known that a nonspecific inhibitory effect is efficiently exhibited by modifying a polymer such as polyethylene glycol (PEG) to a peptide which is a lower molecule than Fab' and has binding property to a nonspecific reaction substance. Although Fab' is a molecule having a molecular weight of about 55kDa, according to Japanese patent No. 5189098, the nonspecific inhibitory effect is lost even when the molecular weight is such a level. However, the inventors have found the following surprising effects: even though the peptide is extremely small of about 1kDa, the peptide can be conjugated with PEG of about 5kDa, and the molecular weight is smaller than that of Fab', and a high nonspecific inhibitory effect can be obtained.
The technical problems of the invention can be solved by the invention as follows:
[1] a non-specific reaction inhibitor for use in an immunological assay comprising a complex of a peptide or nucleic acid molecule and a polymeric compound, the peptide or nucleic acid molecule specifically binding to a non-specific reactive substance.
[2] The nonspecific reaction inhibitor according to [1], wherein the substance specifically binding to the nonspecific reaction substance is a substance capable of chemical synthesis.
[3] The non-specific reaction inhibitor according to [1] or [2], wherein the complex is 50kDa or less.
[4] The non-specific reaction inhibitor according to any one of [1] to [3], wherein the polymer compound is polyethylene glycol.
[5] The non-specific reaction inhibitor according to any one of [1] to [4], wherein the non-specific reaction substance is an immunoglobulin.
[6] An immunological assay method using a complex of a peptide or nucleic acid molecule which specifically binds to a nonspecific reaction substance and a polymer compound.
[7] The immunoassay according to [6], wherein the immunoassay is a latex agglutination optical assay, an immunoscattering turbidimetry, an immunotransmission turbidimetry, a chemiluminescent immunoassay, an enzyme immunoassay, a fluorescent immunoassay or a radioimmunoassay.
[8] An immunoassay reagent comprising a complex of a peptide or nucleic acid molecule that specifically binds to a non-specific reactive substance and a polymeric compound.
[9] The immunological measurement method according to [8], wherein the stabilizing reagent comprises a complex of a peptide or nucleic acid molecule and a polymer compound, and the peptide or nucleic acid molecule specifically binds to the nonspecific reaction substance.
(III) beneficial effects
According to the present invention, since the cost is cheaper than that of biological materials and chemical synthesis is possible, the lot difference can be reduced. Further, according to the present invention, since the polymer has a lower molecular weight, the polymer has high solubility, and a sufficient amount of the polymer can be ensured.
Drawings
Fig. 1 is an explanatory diagram for explaining each step of measurement by using Octet (trademark) R2.
Detailed Description
The nonspecific reaction inhibitor of the present invention comprises a complex of a peptide or nucleic acid molecule (hereinafter, sometimes referred to as binding partner) that specifically binds to a nonspecific reaction substance and a polymer compound.
The term "nonspecific reaction substance" as used herein refers to a substance that causes a nonspecific reaction in an immunological measurement method using an antigen-antibody reaction. Specific factors include human IgM, human IgG, human IgA, human IgE, human IgD, and factors that bind to the antibody when a human body fluid is used as a sample, for example, complement, rheumatoid factor, fc receptor, and the like, and IgM, igG, igA, igE of an animal other than a human body and factors that bind to the antibody when a body fluid of the animal other than a human body is used as a sample.
Examples of the immunological measurement method include latex agglutination optical measurement, immunoscattering turbidimetry, immunotransmission turbidimetry, chemiluminescent immunoassay, enzyme immunoassay, fluorescent immunoassay, and radioimmunoassay. The latex agglutination optical assay, the immunonephelometry, or the other assays without a B/F separation step are particularly susceptible to the effects of nonspecific reaction substances. In such a measurement method, the concentration of a test sample in an antigen-antibody reaction is about several% in many cases, and a larger amount of a nonspecific reaction inhibitor needs to be added as the concentration of the test sample is higher. Even if the concentration of the sample to be measured in the reaction solution is higher than the conventional high concentration (4% or more, 5% or more, or 8% or more), the nonspecific reaction inhibitor of the present invention can ensure a sufficient amount of the sample to be added. Although antigen-antibody reactions are used, among antibodies that detect target antigens, polyclonal antibodies and monoclonal antibodies exist. In any measurement method, the non-specific reaction inhibitor of the present invention may be added to the sample to be measured before or during the antigen-antibody reaction, and is preferably added before the antigen-antibody reaction.
The binding partner that specifically binds to the nonspecific reaction substance used in the present invention includes a peptide or a nucleic acid molecule (e.g., an aptamer), but in the present invention, an antibody or an antibody fragment of Fab' or more is not used. Typically, the molecular weight of the binding partner is 0.5 to 47kDa, preferably 0.8 to 15kDa, more preferably 1 to 8kDa. In the present invention, the use of a binding partner having a molecular weight smaller than that of Fab' can improve the solubility and ensure a sufficient amount of the binding partner to be added. According to the present invention, since the amount of the non-specific reaction inhibitor added to the reaction system in which the immunocomplex formation is performed can be increased, as a result, a sample to be tested having a high concentration of the non-specific reaction substance can be handled, and the concentration of the sample to be tested in the reaction system can be increased.
As the binding partner used in the present invention, a substance capable of chemical synthesis is preferable. In the present invention, since an antibody or an antibody fragment is not used, a low-cost and low-lot-difference nonspecific reaction inhibitor can be provided. The binding partner may suitably contain a functional group for modifying the polymer compound, biotin, a spacer (spacer), or the like.
Examples of the non-specific reaction inhibitor (i.e., a complex between a binding partner of a non-specific reaction substance and a polymer compound) of the present invention include a chemically modified substance obtained by chemically modifying a peptide or a nucleic acid molecule with a polymer compound, and a complex between a peptide or a nucleic acid molecule bound by biotin-avidin binding and a polymer compound.
In the chemical modification, for example, thiol, amino, hydroxyl, or carboxyl groups can be targeted for binding via a "reactive derivative".
The "reactive derivative" used for the thiol group-targeting modification includes, for example, thiol group-selective reactive groups such as maleimides and vinyl sulfones. In addition, a substance obtained by directly bonding a reactive derivative to a polymer may be used, or a crosslinking agent containing a reactive derivative may be used.
The "reactive derivative" used for the amino group-targeting modification includes, for example, N-hydroxysuccinimide (NHS) ester, N-hydroxysulfosuccinimide (Sulfo-NHS) ester, and the like. In addition, there are compounds containing aldehyde groups (for example, glutaraldehyde), polymers containing aldehyde groups in advance, which are polymers, and the like.
The modification targeting a carboxyl group includes, for example, a complex obtained by reacting a carbodiimide (1-Ethyl-3- [3-dimethylaminopropyl ] carbodiimide hydrochloride (1-Ethyl-3- [3-dimethylaminopropyl ] carbodiimidehydrochloride)) with an amino group as a catalyst.
In the modification targeting a hydroxyl group, for example, a compound containing an isocyanate derivative can be used for the preparation.
The polymer into which the reactive derivative is introduced may be obtained as a commercially available product (for example, NOF CORPORATION), or may be prepared by a usual chemical method.
Furthermore, the binding form of the binding partner to the polymer is not based on covalent bonds like biotin and avidin, but methods using binding forms with high affinity are also comprised in the present invention.
Examples of the polymer compound that can be used in the present invention include polysaccharides, proteins, and organic polymers.
Examples of the polysaccharides include dextran, dextrin, agarose, carboxymethyl (CM) cellulose, heparin, and soluble starch. The polysaccharide may be linear or branched. For modification with polysaccharides, conventionally known periodic acid oxidation method, cyanogen bromide method, carbodiimide method, cyanuric chloride method, epichlorohydrin method, SPDP (N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionic acid (N-Succinimidyle 3- [2-pyridyldithio ] propionate)) reagent method, active ester method, and the like can be used. The polysaccharide having the reactive derivative introduced therein may be obtained as a commercially available product or may be prepared by a usual chemical method.
The protein is a complex formed by binding a plurality of amino acids via peptide bonds, and may be purified by animals, artificially prepared by genetic engineering, or prepared by chemical synthesis like a synthetic peptide. Among proteins, casein, milk casein, gelatin, recombinant albumin, and the like are included, for example. The polyamino acid includes homopolymers such as arginine, lysine and glutamic acid; random polymers of lysine with glycine, lysine with serine, and the like. As examples of the binding method of proteins, there are the following methods: the crosslinking agent is bound to the amino group, carboxyl group, thioether group, or the like of the protein as a target, and is bound to the binding partner via the crosslinking agent. In addition, the following methods exist: the carbodiimide is used as a catalyst to bind the binding partner to the protein. In the present invention, the following method is preferred: allowing EMCS [6- (maleimido) hexanoic acid succinimidyl ester (N- (6-Maleimidocaproyloxy) succininimide); DOJINDO Co.) or SMCC [4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (succinimdyl 4- (N-maleimidomethyl) cyclohexane carbonate); a crosslinking agent such as DOJINDO Co., ltd.) is reacted with an amino group of a protein, and the crosslinking agent is bound to a binding partner having a thioether group introduced at the terminal. The protein into which the reactive derivative is introduced, which is used in the production of the product of the present invention, may be obtained as a commercially available product, or may be produced by a general chemical method.
Examples of the organic polymer include polyethylene glycol, polyvinyl alcohol, polyallylamine, polyethylenimine, polymethyl methacrylate, polyacrylic acid, polyallylamine, and polysaccharide. The organic high molecular polymer may be linear or branched, or may be a plurality of random copolymers. The polymer may be a synthetic polymer having a spherical structure such as dendrimer (dendrimer). The polymer may be a synthetic or naturally occurring polymer.
Polyethylene glycol is a polymer compound with a structure formed by polymerizing ethylene glycol as a basic component. Other functional groups can be introduced into the hydroxyl group of polyethylene glycol, and the antibody can be bound by using the functional groups. Examples of the activation performed when the polyethylene glycol is bound to the antibody include methods using cyanuric chloride, carbonyldiimidazole, N-hydroxysuccinimide, and carbodiimide. As the polyethylene glycol into which the functional group is introduced, commercially available polyethylene glycol can be used. When commercial products in which a maleimide group, a succinimide group, an amino group, a thioether group, or the like is introduced into polyethylene glycol are used, the preparation can be efficiently performed. The polyethylene glycol having a maleimide group or a succinimido group introduced therein is more preferable because it has a favorable binding efficiency with a binding partner having a thioether group or an amino group at the terminal. The polyethylene glycol may be linear or branched, and may be substituted with a part of other chemical structure, or modified with other polymer or compound.
As a method of chemical modification using an organic polymer other than polyethylene glycol, there is a method of introducing a functional group into an organic polymer and binding it to an antibody, similar to the above-described method of chemical modification of polyethylene glycol. In addition, in the case of an organic high molecular polymer already containing a reactive derivative, there is a case where it is not necessary to introduce a new functional group into the organic high molecular polymer in the production stage. In the present invention, a polymer having maleimide groups, succinimidyl groups, amino groups and carboxyl groups introduced therein is preferably used. The organic high molecular polymer into which the reactive derivative is introduced may be obtained as a commercially available product or may be prepared by a usual chemical method.
The molecular size of the polymer compound is not particularly limited, but the average molecular weight is usually about 200Da to 1000kDa, for example, 1kDa to 1000kDa, preferably 10kDa to 100kDa. In the case of polyethylene glycol, it is preferably 3kDa to 49.5kDa. The molecular size can be appropriately selected in consideration of hydrophilicity, steric structure, nonspecific inhibitory effect, and the like, depending on the kind of polymer used.
The nonspecific reaction inhibitor of the present invention can be used by adding a complex of a binding partner as an active ingredient thereof and a polymer compound to an immunoassay system. Specifically, a solution of a binding partner modified with a polymer compound may be prepared, and the above-mentioned solution may be added to a sample in advance before an antibody against an antigen to be measured is reacted with the antigen, so that a non-specific reaction substance is reacted with the binding partner against the non-specific reaction substance, thereby suppressing a non-specific reaction by the non-specific reaction substance; alternatively, a binding partner modified with a polymer compound may be contained in a solution of an antibody against an antigen to be measured, and the solution may be added to a sample to react a non-specific reaction substance with the binding partner against the non-specific reaction substance, thereby suppressing a non-specific reaction by the non-specific reaction substance.
The immunological measurement reagent of the present invention may be constituted by one part or two or more parts, as long as the nonspecific reaction inhibitor of the present invention is further contained in a conventional immunological measurement reagent using an antigen-antibody reaction.
When it is constituted of a part, it is constituted of a reactive agent containing at least a support carrying an antigen or an antibody for forming an immunological complex. The signal is detected by reacting the sample with the reagent according to a known method. The nonspecific reaction inhibitor of the present invention may be added to the reagent before the reaction of the sample with the reagent, but is preferably supplied in a state of being added to the reagent. When it is composed of two or more parts, it is composed of a stabilizing reagent and a reactive reagent containing at least an antibody or an antigen-supporting body, which is used to form an immunological complex. The stabilizing reagent may be a reagent for diluting a sample to an appropriate concentration or a reagent for pretreatment, and may be prepared by a known method. The signal is detected by reacting the sample with a stabilizing reagent and then with a reagent according to a known method. The nonspecific reaction inhibitor of the present invention may be added to the stabilizing reagent and/or the reaction reagent before the reaction of the sample with the reaction reagent, but is preferably supplied in a state of being added to the stabilizing reagent and/or the reaction reagent. It is particularly preferable to supply the stabilizing reagent in a state where it has been added thereto.
Examples of measurement items as an immunological measurement reagent include elastase, cystatin C, sEs (soluble E-selectin), SF (soluble fibrin), PC (protein C), PPI (plasmin inhibitor), cTn (thrombomodulin), myoglobin, CK-MB, BNP (type B natriuretic peptide), NT-proBNP (N-terminal BNP precursor), cTnI (cardiac troponin I), AFP (alpha-fetoprotein), beta 2M (beta-2-microglobulin), CEA (carcinoembryonic antigen), ferritin, CA19-9 (saccharide antigen 19-9), PAP (prostaacid phosphatase), PSA (prostate-specific antigen), CRP (C-reactive protein), mb (myoglobin), PCT (procalcitonin), sCD14 subtype (presepsin), IL-2R (interleukin-2 receptor), RF (rheumatoid factor), ASO (anti-streptococcal protein-O), FDP (fibrinolytic product), ATIII (thrombin III), thrombin-gamma-fetoprotein (IgG-2), igA (immunoglobulin G-gamma-fetoprotein), and Ig-2 (immunoglobulin G-D (immunoglobulin-G) fragment-factor, and immunoglobulin-alpha-fetoprotein (immunoglobulin-D-protein-B) fragment-such as a measurement item, igE (immunoglobulin E), C3 (complement third component), C4 (complement fourth component), urinary albumin, hCG (human chorionic gonadotrophin), hPL (human placental lactogen), insulin, HBs antigen (hepatitis B surface antigen), HBs antibody (anti-hepatitis B surface antigen antibody), HBc antibody (anti-hepatitis B core antigen antibody), HCV antibody (anti-hepatitis C virus antibody), treponema (anti-treponema pallidum antibody), TSH (thyroid stimulating hormone), LH (luteinizing hormone), FSH (follicle stimulating hormone), prolactin, testosterone, estradiol, progesterone, digoxin, digitoxin, quinidine, procaine, NAPA (N-acetyl procaine), theophylline, phenytoin, phenobarbital, carbamazepine, valproic acid, ethosuximide, gentamicin, buxine, amicin, lincomycin, cyclosporine A, B (vitamin B12), T3, tri-iodo thyroxine (thyroxine), thyroxine (T4).
Examples
As a small molecule to which a non-specific reactive substance is bound, a chemically synthesized peptide or nucleic acid aptamer is used, and as a polymer compound to modify the chemically synthesized peptide or nucleic acid aptamer, polyethylene glycol (hereinafter, referred to as PEG) is used. The present invention will be specifically described below by way of examples, which are not intended to limit the scope of the present invention.
Example 1: preparation of PEG-modified peptides
One molecule of PEG is conjugated to one molecule of peptide via the N-terminal amino group. For the peptide, a human IgG-binding peptide of about 1.5kDa (amino acid sequence: DCAWHLGELVWCT: SEQ ID NO: 1) known for the purpose of purification of human IgG and a human IgA-binding peptide of about 1.8kDa (amino acid sequence: HMVCLSYRGRPVCFSL: SEQ ID NO: 2) reported for the purpose of purification of human IgA were used, and PEG was produced using PEG having a succinimidyl group at one terminal (molecular weight 20kDa;NOF CORPORATION).
[ Preparation of PEG-modified IgG binding peptides ]
After dissolving the human IgG-binding peptide in 0.2mol/L NaHCO 3 (pH 8.5) so that the concentration of the peptide became 1.0mg/mL, PEG having a succinimidyl group at one terminal was added in an equimolar amount to the peptide, and the mixture was stirred using a rotator and reacted overnight at 4 ℃. After the reaction, phosphate Buffered Saline (PBS) was used as running buffer, and the reaction mixture was passed through a human IgG affinity column to remove unreacted PEG. Elution was performed using 0.2mol/L citrate buffer (pH 3.0), concentrated by ultrafiltration and unreacted peptide removed, and the buffer replaced with PBS. Concentration measurement was performed by a quantitative colorimetric peptide detection kit (Quantitative Colorimetric PEPTIDE ASSAY) (Thermo FISHER SCIENTIFIC k.k.manufactured), and stored at 4 ℃.
[ Preparation of PEG-modified IgA-binding peptide ]
The human IgA-bound peptide was dissolved in PBS (pH 7.4) containing 40% dimethyl sulfoxide (DMSO) so that the concentration of the peptide became 1.0mg/mL, and then PEG having a succinimide group at one terminal was added in an equimolar amount to the peptide, followed by stirring with a rotator and overnight reaction at 4 ℃. After the reaction, PBS was used as a running buffer and passed through a human IgG affinity column to remove unreacted PEG. Elution was performed using 0.2mol/L citrate buffer (pH 3.0), concentrated by ultrafiltration and unreacted peptide removed, and the buffer replaced with PBS. Concentration measurement was performed by a quantitative colorimetric peptide detection kit (Thermo FISHER SCIENTIFIC k.k., manufactured) and stored at 4 ℃.
Example 2: confirming the effect of inhibition of the reaction with human IgG or human IgA when PEG modification was performed and when PEG modification was not performed
As a model system for confirming a nonspecific inhibitory effect, an anti-human IgG antibody and human IgG or an anti-human IgA antibody and human IgA were used, and the effect of the inhibition reaction generated when PEG modification was performed or not performed was confirmed on an IgG-binding peptide or an IgA-binding peptide having a molecular weight smaller than that of the antibody fragment.
[ Method ]
The measurement was performed by a biological film interferometry (Bio-Layer Interferometry:BLI) using Octet (trademark) R2 (manufactured by Sidoris Co., ltd.) as a biological molecule interaction analysis system. The basic principle of the biological film interferometry is briefly described. When light of a specific wavelength is projected onto a bio-molecular layer (film) fixed on the surface of the sensor tip, the light is reflected at both surfaces of the bio-molecular film and the film as an internal reference, generating interference waves of light. When molecules in the measurement sample bind to biomolecules on the surface of the sensor tip, the thickness of the film of the sensor tip increases, and the interference wave is shifted in wavelength. By measuring the change in the wavelength shift, the number of molecules bound to the biomolecules immobilized on the sensor tip surface can be quantified and dynamically analyzed in real time. In this example, a sample to be tested was allowed to coexist with a nonspecific reaction inhibitor in a measurement sample, and human IgG or human IgA was absorbed in a sample solution. The determination is carried out according to the instructions attached OctetR 2.
[ Confirmation of the Effect of inhibiting the reaction with human IgG ]
As an antibody solution, an anti-human IgG polyclonal rabbit antibody (manufactured by DANCE Co., ltd.) was diluted to 25. Mu.g/mL with PBS (-) to prepare an anti-human IgG antibody solution. The sample solution was used as a solution prepared in the following manner: to 20. Mu.L of a sample to be tested, 1mg/mL of human IgG was prepared using PBS (-), and to the sample to be tested, 20. Mu.L of the IgG-binding peptide or the 20 kDa-modified PEG was added so that the final concentration was 25. Mu.g/mL or 50. Mu.g/mL, and the total amount was 200. Mu.L using LPIA GENESIS TAT (manufactured by LSI MEDIENCE CORPORATION) of the first reagent (hereinafter, referred to as R1) as a thrombin-antithrombin III complex (hereinafter, referred to as TAT) measuring reagent.
A sample solution and an anti-human IgG antibody solution were added to a predetermined well of a 96-well black well plate (manufactured by Greiner Bio-One International GmbH) at 200. Mu.L/well, respectively. 200. Mu.L/well of PBS (-) was added to each of the wells for baseline (baseline), dissociation, and washing. The well plate and a Biosensor (Biosensor/ProL: manufactured by Sidoris Co., ltd.) were placed at a predetermined position OctetR. After OctetR is started up and data is obtained under the conditions shown in table 1 below, the value of the change in wavelength shift (Response) is calculated using analysis software attached to OctetR 2. In addition, the measurement was carried out at a temperature of 30 ℃.
TABLE 1
[ Confirmation of Effect of inhibiting reaction with human IgA ]
As an antibody solution, an anti-human IgA polyclonal rabbit antibody (manufactured by DANCO Co., ltd.) was diluted to 25. Mu.g/mL with PBS (-) to prepare an anti-human IgA antibody solution. The sample solution was used as a solution prepared in the following manner: human IgA prepared at 250. Mu.g/mL using PBS (-) was used as a test sample, and to 20. Mu.L of the test sample, igG-binding peptide or 20 kDa-modified PEG-IgA-binding peptide was added so that the final concentration was 25. Mu.g/mL or 50. Mu.g/mL, and the total amount was 200. Mu.L using R1.
The measurement using OctetR2 was performed in the same manner as "confirmation of the effect of suppressing the reaction with human IgG".
Results (results)
The effect of suppressing the reaction with human IgG is shown in table 2, and the effect of suppressing the reaction with human IgA is shown in table 3. By modifying PEG, the effect of the IgG-binding peptide or the IgA-binding peptide to inhibit the reaction of the anti-human IgG antibody with human IgG or the reaction of the anti-human IgA antibody with human IgA is increased as compared to the case where no modification is performed. It was found that IgG-binding peptides or IgA-binding peptides were 1 to 2kDa, and showed an effect of suppressing the reaction even when the molecular weight of 20kDa PEG was 21 to 22kDa, i.e., the molecular weight was smaller than that of the antibody fragment.
The reason why the measurement value increases by the addition of the peptide of unmodified PEG is considered as follows: the peptide binds to human IgG or human IgA, but does not generate sufficient steric hindrance to inhibit the binding of the anti-human IgG antibody to human IgG or the binding of the anti-human IgA antibody to human IgA, the human IgG to which the IgG-binding peptide is bound to the anti-human IgG antibody, and the human IgA to which the IgA-binding peptide is bound to the anti-human IgA antibody, so that the thickness of the membrane at the sensor tip increases and the change in wavelength shift increases as compared to the case where only human IgG or human IgA binds to the anti-human IgG antibody or the anti-human IgA antibody without the addition of the non-specific reaction inhibitor.
TABLE 2
TABLE 3
Example 3: confirm the effect of inhibiting the nonspecific reaction generated when PEG modification was performed and when PEG modification was not performed ]
The effect of inhibiting the nonspecific reaction was confirmed when PEG modification was performed and when PEG modification was not performed on IgG-binding peptides having a molecular weight smaller than that of the antibody fragments, using human plasma showing the nonspecific reaction.
[ Method ]
As the sample to be tested, sample A was used, which was human plasma showing a nonspecific reaction in LPIA GENESIS TAT (manufactured by LSI MEDIENCE CORPORATION), and the specific reaction substance was IgG.
The measurement was performed in the same manner as the measurement conditions described in example 2. In this example, in the measurement sample, sample a was allowed to coexist with a nonspecific reaction inhibitor, and a nonspecific reaction substance was absorbed in a sample solution.
As the antibody solution, an anti-TAT antibody solution was used, which was obtained by diluting an anti-TAT antibody used in a LPIA GENESIS TAT second reagent (hereinafter referred to as R2) to 25. Mu.g/mL with PBS (-), and which was characterized by containing latex particles obtained by immobilizing a monoclonal antibody having a specific binding energy for TAT. The sample solution was used as a solution prepared in the following manner: to 20. Mu.L of the sample to be tested diluted 10-fold with PBS (-), an IgG-binding peptide as a nonspecific reaction inhibitor or an IgG-binding peptide modified with 20kDa PEG was added so that the final concentration was 25. Mu.g/mL or 50. Mu.g/mL, and R1 was used to make the total amount 200. Mu.L.
Results (results)
The results are shown in Table 4. As with the results of example 2, the inhibition effect of the nonspecific reaction of IgG-binding peptide was increased by modifying PEG as compared to the case where no modification was performed. In addition, the PEG-modified IgG-binding peptide, although having a smaller molecular weight than the antibody fragment, exhibits a nonspecific reaction inhibition effect.
The reason why the measurement value increases by the addition of the peptide of unmodified PEG is considered as follows: the IgG-binding peptide binds to the non-specific reaction substance, but does not generate steric hindrance enough to inhibit the binding of the non-specific reaction substance to the anti-TAT antibody, and the non-specific reaction substance to which the IgG-binding peptide is bound binds to the anti-TAT antibody, so that the thickness of the membrane at the sensor tip is increased and the change in wavelength shift is larger than that in the case where only the non-specific reaction substance binds to the anti-TAT antibody when the non-specific reaction inhibitor is not added.
TABLE 4
Example 4: comparison of the effects of PEG-modified peptides and antibody fragments
From the results of example 2 and example 3, it was found that the PEG-modified peptides showed a nonspecific reaction inhibition effect although the molecular weight was smaller than that of the antibody fragments. It can be said that this is a new finding that overtakes the report of "the effect of suppressing the nonspecific reaction is small because the molecular weight of the antibody fragment is small, and the effect of suppressing the nonspecific reaction is exhibited by modifying PEG to make it huge" in japanese patent No. 5189098. Therefore, the effect of suppressing the nonspecific reaction in the case of PEG modification of the peptide bound to the nonspecific reaction substance was compared with the effect of suppressing the nonspecific reaction in the case of PEG modification of the antibody fragment bound to the nonspecific reaction substance.
[ Method ]
PEG modified antibody fragments chemically modified with 20kDa PEG containing maleimide groups at the ends were prepared using Fab' reacted with human IgG. After pepsin digestion of goat anti-human IgG polyclonal IgG (purified from antiserum manufactured by International Immunology Corporation) to prepare F (ab '), fab' was further prepared using 0.1mol/L TCEP-HCL, which was reduced at 37 ℃ for 210 minutes. To 5mg/mL of the Fab' solution, 20kDa PEG (manufactured by NOF CORPORATION) conjugated with maleimide groups was added, and the mixture was reacted at 37℃for 30 minutes. The reaction solution was concentrated to about 5mg/mL by ultrafiltration, and the buffer was replaced with 0.2mol/L TBS to obtain PEG-modified anti-human IgG Fab'.
The non-specific reaction inhibition effect of the PEG-modified peptides described in examples 2 to 3 and the prepared PEG-modified antibody fragments was compared by the same method as the measurement conditions described in example 3. The same sample A as in example 3 was used as a test sample, except that the amount of the non-specific reaction inhibitor to be added was 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL or 400. Mu.g/mL.
Results (results)
The results are shown in tables 5 and 6. When the addition amounts are made equal, the nonspecific reaction inhibition effect of the PEG-modified peptide is higher than that of the PEG-modified antibody fragment. Thus, it was revealed that the PEG-modified peptide had a sufficient nonspecific reaction suppressing effect even in a small amount.
TABLE 5
TABLE 6
Example 5: confirm the nonspecific reaction inhibition effect when the molecular weight of the modified PEG was increased (I)
In Japanese patent No. 5189098, it was confirmed that the greater the molecular weight of PEG of the modified antibody fragment, the more the nonspecific reaction suppressing effect was enhanced. Therefore, the nonspecific reaction inhibition effect of PEG-modified peptides upon changing the molecular weight of the modified PEG was confirmed.
[ Method ]
A PEG-modified IgG-binding peptide chemically modified with 5kDa, 10kDa, 20kDa, 30kDa PEG having a succinimidyl group at the terminal was prepared by the method described in example 1.
[ Measurement conditions for evaluating the Effect of nonspecific reaction inhibitor ]
The same measurement conditions as those described in example 3 were used. As a non-specific reaction inhibitor, a sample solution was prepared to which a PEG-modified peptide was added, in which PEG of 5kDa, 10kDa, 20kDa, and 30kDa was chemically modified, and the non-specific reaction inhibition effect was confirmed. The amount of the nonspecific reaction inhibitor added was 5. Mu.g/mL, 10. Mu.g/mL, or 20. Mu.g/mL, and the same sample A as in examples 3 and 4 was used as the test sample.
Results (results)
The results are shown in Table 7. The PEG modified with any molecular weight shows a nonspecific reaction inhibition effect. Further, it was confirmed that the effect became high depending on the molecular weight of PEG.
TABLE 7
Example 6: confirm the nonspecific inhibition effect when using an automatic analyzer
An automatic analyzer is actually used to confirm the nonspecific inhibitory effect when measured by the latex agglutination method.
[ Method ]
The nonspecific inhibitory effect of the IgG-binding peptide and the PEG-modified IgG-binding peptide used in example 2 was confirmed by the latex agglutination method.
The measurement was performed by an automated operation of an automatic analyzer STACIA (manufactured by LSI MEDIENCE CORPORATION). In the measurement performed with STACIA, two operations were mainly performed. In the first operation, the sample to be measured was diluted with R1 and heated at 37℃for 3.5 minutes. In the second operation, R2 was added to the reaction solution, and the mixture was heated at 37℃for 6 minutes to cause latex agglutination. By optically monitoring the agglutination reaction, TAT or a nonspecific reaction substance in a non-test sample is quantified. In this example, a nonspecific reaction inhibitor was added to R1 so that the concentration after the addition of the sample was 0.05mg/mL or 0.1mg/mL, and the nonspecific reaction substance was absorbed in the first operation. The same amount of PBS (-) was added to R1 without the non-specific reaction inhibitor, avoiding the effect of the additive on dilution. The mixing ratio of the measurement sample, R1 and R2 was set to 20. Mu.L/90. Mu.L. The latex was examined for aggregation at a wavelength of 700 nm. For the measured value, the measured value was calculated by comparing absorbance based on a calibration curve obtained by measuring TAT at a known concentration. The same test sample A as in examples 3 to 5 was used as the test sample.
Results (results)
The results are shown in Table 8. In the latex agglutination method, the nonspecific inhibitory effect by the PEG-modified IgG-binding peptide was also confirmed. The reason why the measurement value increases by the addition of the peptide of unmodified PEG is considered as follows: the IgG-binding peptide binds to the non-specific reaction substance, but does not generate sufficient steric hindrance to inhibit the binding of the non-specific reaction substance to the anti-TAT antibody, and the non-specific reaction substance to which the IgG-binding peptide is bound binds to the anti-TAT antibody, so that absorbance changes due to an increase in the size or complexity of the aggregate, as compared to the case where only the non-specific reaction substance binds to the anti-TAT antibody when the non-specific reaction inhibitor is not added.
TABLE 8
Example 7: confirming the effect of inhibiting the reaction between an anti-human IgG antibody and human IgG when the IgG-binding aptamer was modified with or without PEG
The effect of the inhibition reaction between the PEG modification and the non-PEG modification was confirmed for the nucleic acid aptamer by using a model system for confirming the nonspecific inhibition effect in the same manner as in example 2.
[ PEG-modified IgG-binding nucleic acid aptamer ]
Nucleic acid aptamer using a human IgG-binding nucleic acid aptamer of about 7.5kDa that has been reported for purification of human IgG (nucleic acid sequence: N (6) _ggaggu (F) GcU (F) CCGAAAGGA (L) aC (F) U (F) cc (capital = RNA, lowercase = DNA, N (F) = 2 '-fluoro RNA, N (L) = LNA, N (6) = Amino C6 Linker (Amino C6 Linker)). PEG-modified nucleic acid aptamer using an aptamer in which one molecule of PEG is bound to one molecule of nucleic acid aptamer via an Amino group at the N-terminus (nucleic acid sequence: p_n (6) _ggaggu (F) GcU (F) CCGAAAGGA (L) aC (F) U (F) cc (capital = RNA, lowercase = DNA, N (F) 2' -fluoro RNA, N (L) = LNA, N (6) = Amino C6 Linker, p=20 kDa)).
[ Method ]
The test sample and the measurement were carried out under the same measurement conditions as in example 2. In this example, a sample to be tested was allowed to coexist with a nonspecific reaction inhibitor in a measurement sample, and human IgG was absorbed in a sample solution. The determination is carried out according to the instructions attached OctetR 2.
As an antibody solution, an anti-human IgG polyclonal rabbit antibody (manufactured by DANCE Co., ltd.) was diluted to 25. Mu.g/mL with PBS (-) to prepare an anti-human IgG antibody solution. The sample solution was used as a solution prepared in the following manner: a total of 200. Mu.L was prepared by adding 20. Mu.L of a sample to be tested, which was prepared by using PBS (-) to 1mg/mL of human IgG as a sample to be tested, and adding 20. Mu.L of the sample to the IgG-binding aptamer or the 20 kDa-modified PEG to give a final concentration of 0.3mg/mL, using a reaction buffer (145 mmol/L NaCl,5.4mmol/L KCl,0.8mmol/L MgCl 2, 1.8mmol/L CaCl 2, 20mmol/L Tris (pH 7.6), 0.05% Tween 20).
Results (results)
The results are shown in Table 9. By modifying PEG, the effect of IgG-binding aptamer in inhibiting the reaction of anti-human IgG antibody with human IgG is increased compared to the case where no modification is performed. In addition, the IgG-binding aptamer was about 7.5kDa, and showed an inhibitory effect on the reaction even when the molecular weight of 20kDa PEG was 27.5kDa, i.e., the molecular weight was smaller than that of the antibody fragment.
TABLE 9
Industrial applicability
The nonspecific reaction inhibitor of the present invention can be suitably used for immunological measurement.
Sequence listing of independent text
The amino acid sequences represented by the sequences SEQ ID NO. 1 and SEQ ID NO. 2 of the sequence list are respectively human IgG binding peptide and human IgA binding peptide.
Claims (9)
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| JP4065600B2 (en) | 1998-04-01 | 2008-03-26 | デンカ生研株式会社 | Immunological assay and immunological assay kit |
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